Western blot semiquantitative analysis of noncanonical campdependent protein expression induced by pacap emily jones1, yvonne holighaus2, and lee eiden2 1montgomery blair high school 2national institutes of health abstract the neuropeptide pituitary adenylate cyclase activating polypeptide pacap is a slow transmitter and a. Comparative analysis of erk phosphorylation suggests a mixed. Aug 06, 2014 this is a short tutorial on how to remove the background, rotate and finally analyze your scanned western blot image. Following separation of the biological sample with sds page and subsequent transfer to a membrane usually pvdf or nitrocellulose, a phosphospecific antibody can be used to identify the protein of interest view phosphospecific antibodies for western blot analysis. Western blot analysis the following antibodies were used for western blot analysis in supplementary figure 3. Cells were stimulated for 5 minutes with 100 ngml egf and 1. Reversal of multidrug resistance by gefitinib via raf1erk. Total erk and p38 levels were determined from antierk and. Nc104 cells at pd 18 were approaching permanent growth arrest pd20, which was included to show upregulation of p16 ink4a for the comparison with p16 ink4a levels after hpv 16 e6e7 expression. Neuronal extracts were prepared and analyzed by western blot using ras c, perk e, total erk g, and cyclin d1 h antibodies.
Four samples of erk in different states of phosphorylation were prepared as follows. Hrp conjugated antibodies were used as the secondary antibodies. Western blot semiquantitative analysis of noncanonical. Gs 700 imaging densitometer and the molecular analyst software. How should we analyze the two bands of phospho erk12 in. After washing the membranes, the reaction was visualized using the ecl detection kit ge healthcare, buckinghamshire, uk and by exposing the blots to fuji medical xray film. A guide to modern quantitative fluorescent western blotting. The results of western blot were analyzed with the analysis software quantity one biorad, hercules, ca. Sophoridine induces apoptosis and s phase arrest via ros.
D western blot analysis of fbw7 expression in panc1 and sw1990 cells in the presence and absence of the. The size of erk12 is around 45 kda therefore a 8% page is sufficient to visualize the two bands. Affinityprecipitated erk and perk were detected on western blots. The kinetic data of the interaction were evaluated with scrubber2 biologic software. Western blot analysis of pi3kakt and rasrafmapk signalling. D nuclear and cytoplasmic extracts were isolated from mcf7 neo and mcf7 snail cells. To evaluate the erk12 signaling pathway, we focused on ras, mek12 and erk12 proteins and assessed the abundance of these and their phosphorylated isoforms in the brains of btbr and b6 mice using western blot analysis figure 2. Miapaca2 and panc1 cancer cells were treated with sophoridine at different concentrations for 48 h, and then wholecell lysates were prepared for western blot analysis as previously described.
Expression of yaphippo pathway and cell viability analysis after erk inhibition in nsclc cells. All dedicated western blot analysis software from major suppliers. Gpcrs gprotein coupled receptors activate the mapk cascade using a different set of adaptors. Aug 24, 2009 the perk protein is a key downstream component of the mek erk cascade. Similarly, we found that the mekspecific inhibitor u0126 exerted a sensitization effect following irradiation because mek is upstream of erk signaling. Proportional cell viability was analyzed with graphpad prism6 software graphpad software. Sep 14, 2016 similarly, we found that the mekspecific inhibitor u0126 exerted a sensitization effect following irradiation because mek is upstream of erk signaling. Analysis of the bands of interest depends on the type of detection and the imaging system available, with many imaging systems requiring proprietary software for image acquisition and quantification. C western blot analysis of wt and dtg mice hearts for 6xhis tagged proteins, total and phosphorylated tey or t207 erk1 levels of whole. Many optimized western blotting methodologies exist and are utilized in different laboratories.
Boster recommends enhanced chemiluminescent kit with antimouse igg ek1001 for western blot. Quantitate western blot data using imaging systems software or a preferred standalone software if available. Comparative analysis of erk phosphorylation suggests a. Mar 12, 2010 to be sure about p44p42 phosphorylated or not, treat blot membrane with a antierk12 antibody. A guide to modern quantitative fluorescent western. Designed for the analysis of nearinfrared western blot images acquired on an odyssey imager, empiria studio is the only western blot analysis software that guides you all the way from validation to statistical analysis. The expression of snail, perk, erk, pelk1, elk1, pp90rsk, and p90rsk was analyzed by western blot analysis. Western blot comparison western blot assays are a labor intensive, low throughput, timeconsuming, and nonquantitative approach to detecting protein biomarkers. Projects that are difficult or impossible with western blotting. Detecting phosphorylated erk12 sdspage and western blotting. Expression of erk and p erk proteins with western blot analysis.
A systematic approach to quantitative western blot analysis. Expert western blot analysis made simple with empiria studio software. Densitometry data generated for western blots are commonly used to compare. Licor image studio lite free densitometry software. Activation of raserk signaling and gsk3 by amyloid. Erk kinase phosphorylates and destabilizes the tumor. Dual roles of extracellular signalregulated kinase erk in. Erk 12 activation mediates the neuroprotective effect of bpv. This is a quick look at the free densitometry software from licor. Dapi was used in immunofluorescence to identify the nuclei.
Normalization uses an internal loading control to correct for unavoidable sampletosample and lanetolane variations. To evaluate the erk12 signaling pathway, we focused on ras, mek12 and erk12 proteins and assessed the abundance of these and their phosphorylated isoforms in the brains of btbr and b6 mice using western blot analysis. The western blot sometimes called the protein immunoblot, or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a. For more detailed descriptions of western blotting andor icw techniques, refer to western blot analysis and incell western. The beads were removed and depleted lysates were then incubated at 30 c for 20 min prior to western blot analysis ac. Phosphospecific rabbit anti erk mapk thr 202, tyr 204 antibody validated for wb, ihc in human, mouse, rat. Western blotting analysis of yap protein level after erk12 inhibition in nsclc cells.
A regular scanner with a good resolution can also do the job. Accurate, replicable western blots with the odyssey clx. The present study showed that cell proliferation on 3d collagen matrices was significantly less inhibited in endometriotic stromal cells than in endometrial stromal cells. All dedicated western blot analysis software from major suppliers provides the ability to measure o. Densitometric analysis was performed using nih imagej software. The typical western blot protocol eliminates the hazards and waste disposal. Western blot and incell western assay detection with.
Comparison of immunohistochemical and western blot analyses of paraffinembedded cells and xenografts for profiling cellular signaling. Immunofluorescent analysis of phosphop4442 mapkerk12 pthr202tyr204 in nih3t3 cells. Neuronal extracts were prepared and analyzed by western blot using ras c, p erk e, total erk. Western blot analysis using antibodies specific to the phosphorylated forms of erk a, or p38 b for both mockdepleted or vhrdepleted lysates at 0 and 20 min. Structural and functional analysis of phosphorylationspecific. Multiple bands within a western blot raise a critical flag concerning an antibodys specificity. A quantitative image cytometry technique for time series or.
All images are acquired using the licor odyssey classic imager and associated image pro analysis software version 3. Antiphosphomap kinase, activateddiphosphorylated erk1. With this kind of scanner when the minimum image area is selected the scanner minimizes the amount of background software dependant. Pathway analysis with the incell western assay licor. This technical note is a guide to using irdye subclass speci. Fulllength western blots of sirt5 and actin expression in protein extracts. An overview of technical considerations for western blotting applications to physiological research. An overview of technical considerations for western.
Values represented the relative ratio of iba1, bdnf, pi3k, or perk levels normalized to. Dilution series for each of the five antibodies in figure 1c were obtained using the erkx sample with the antix antibody and erkty with antitotal erk over a sample range from 20 to 900 pg supplementary figure 1. Antiphosphotyrosine western blot of h2o2treated cos1 cell extract lane 1, arsenate elution. Erk inhibition promotes neuroectodermal precursor commitment. Extracellular regulated kinases erk 1 and erk2 are authentic. Imaging software is then used to compare the signal generated by the bands detected on the western blot. Western blot analysis of pi3kakt and rasrafmapk sign openi. Dual roles of extracellular signalregulated kinase erk. There is high feasibility to use this software even to quantify cell number and therefore i believe it will assist in studying scratch wound assays. It might well be that your species or the organ produces only one band of the two erkproteins. Puerarin restrains the activation of mek erk 12 pathway. Without western blot normalization, you cant know if changes in band intensity reflect biological change in your samples or variability in sample preparation, loading, and transfer. Accordingly, cpa4 promotes crc cell growth via activating the stat3 and erk pathways and may be a prognostic factor or therapeutic target for crc. Western blot analysis of erk activation in untreated a,c and perk.
Comparison of immunohistochemical and western blot. This is a very nice and free software from licor for quantitative analysis of western blots and dot blots etc. Mapk erk activation in an animal model of social deficits. Image studio lite free western blot quantification. Puerarin inhibits m2 polarization and metastasis of tumor. Densitometers and analysis software in order to make a permanent record, or to get an objective measure of the signal generated on a western blot, a densitometer is used to scan the blot or film. Dec 22, 2014 the erk signaling pathway is upregulated in btbr mice. Helicobacter pylori cytotoxinassociated gene a protein. Moreover, the downregulation of pmek, p erk and bcl2 was consistent with the results obtained in vitro figure 5f. The p erk erk ratio was significantly upregulated by pdgfbb a and tgf. Endostatin inhibits fibrosis by modulating the pdgfrerk. Phosphorylated erk is a potential predictor of sensitivity to. Microglial bdnf, pi3k, and perk in the spinal cord are. Anti phosphotyrosine western blot of h2o2treated cos1 cell extract lane 1, arsenate elution.
Extracellular regulated kinases erk 1 and erk2 are. Automate image acquisition, data analysis and management, and validation. The mapk erk signaling cascade is activated by a wide variety of receptors involved in growth and differentiation including receptor tyrosine kinases rtks, integrins, and ion channels. The levels of phosphorylated and total mek and erk were evaluated by western blot analysis. This is a short tutorial on how to remove the background, rotate and finally analyze your scanned western blot image.
Erk 1, erk 2 antibody produced in rabbit has been used in western blot analysis. Explore biorad image analysis software for 1d and 2d electrophoresis and blotting. The western blot analysis showed aberrant activation of the akt and erk signaling pathways in endometriotic stromal cells on 3d collagen matrices. Western blots were obtained as described below and logfluorescence intensity was plotted against log protein amount. Results western blot analysis showed that the level of phosphorylation of erk in my cell cultures following egf stimulation. Carboxypeptidase a4 promotes cell growth via activating stat3. The mek inhibitors enhance the efficacy of sorafenib.
A the expression of mek, erk 12 and their phosphorylated forms was measured by western blot analysis. Any antibody that produces multiple banding in western blot. Total erk and p38 levels were determined from anti erk and antip38 western analysis and are shown in the lower panels. Currently, several western blot analysis software packages offer. All samples were then subjected to western blot analysis as described above. The western blot analysis showed aberrant activation of the akt and erk. Blocking tbk1 alleviated radiationinduced pulmonary fibrosis. Fluorescence microscopy was performed using zeiss axiovision rel 4. Imagej software was used to analyse the western blot results. Puerarin suppress apoptosis of human osteoblasts via erk. Rabbit polyclonal map kinase erk 1, erk 2 antibody. Cooperation between the akt and erk signaling pathways may.
Dual roles of extracellular signalregulated kinase erk in quinoline compound bpiqinduced apoptosis. Dietary energy restriction inhibits erk but not jnk or p38. Western blot revealed that activation of the stat3 and erk pathways is one of the oncogenic functions of cpa4 in crc. Cordycepin induces apoptosis and g2m phase arrest through. In this study, basal levels of perk were determined by immunocytochemical analysis and western blot analysis in order to evaluate the activation of the rafmek erk pathway in four types of hcc cell lines with different metastatic potential.
The result is a now truly quantifiable fluorescence based western blot qfwb that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Activation of the mapk erk2 is mediated by phosphorylation of a. Western blot of human t47d cell lysate showing specific immunolabeling of 4244 kda erk. Im going to perform a western blot analysis on cell lysate from human. How should we analyze the two bands of phospho erk12 in western blot. Snail promotes epithelial mesenchymal transition in breast. It gives a basic overview of the functionality and i run through the analysis. After phosphoerk was detected, the blot was stripped with restore western blot. The western blot analysis of iba1 a, bdnf b, pi3k c, and perk d proteins in the spinal cord of rats in different groups on d14. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting western blot data in the literature.
Activation of erk1 and erk2 is characterized by a reduced mobility in sdspage as a result of phosphorylation of specific threonine and tyrosine residues. If not, p44a and p42 are probably together in same band. A hcc cell lines bel7402 and smmc7721 were treated with increasing doses 0, 2. Western blot assay was conducted according to a previously published article. Western blot semiquantitative analysis of noncanonical camp. Western blotting, also known as immunoblotting is a core technique in cell and molecular biology. The erk signaling pathway is upregulated in btbr mice. Image lab software suite life science research biorad. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. Western blot of human t47d cell lysate showing specific immunolabeling of 4244 kda erk mapk protein phosphorylated at thr 202 tyr 204 in the first lane.
Among them, western blotting, flow cytometry, and live cell imaging are. Streamlined workflows minimize usertouser variation, compute statistical values, and provide extensive analysis options. Extracellular signalregulated kinase erk activation. Western blotting products include the v3 western workflow, protein transfer systems, blotting membranes, filter paper, premixed blotting. Without the details of your protein isolation, gel % and treatment of the proteins it is impossible to answer your question. Currently, several western blot analysis software packages offer multiple options to subtract background signal and quantify target bands. Apr 15, 2019 c, e western blot analysis of perk, ecadherin, vimentin, and. The peroxidesensitive fluorescent probe dcfhda was used to measure the intracellular ros levels. This may be due to egfrindependent signaling pathways that also signal through erk and were not inhibited by the drug tested. A, western blot analysis using 6e10 antibody shows presence of small a. Gs700 imaging densitometer and the molecular analyst software. Designed for the analysis of nearinfrared western blot images acquired on an odyssey imager, empiria studio is the only western blot analysis software that guides you all the way from validation to statistical analysis for reliable results. Phosphorylation of erk signalling pathway molecules in.
To analyze the effect of erk or estrogen receptor er inhibition on hob apoptosis, hobs were pretreated with 10. Based on the western blot analysis in eca109 cells, cleavedcaspase3, cleavedcaspase9, cleaved parp, and bax all significantly increased. Cooperation between the akt and erk signaling pathways. Our guide provides an overview for performing successful western blots. Normalize western blots to correct for technique variability. The western blot sometimes called the protein immunoblot is a widely used analytical technique in molecular biology, immunogenetics and other molecular biology disciplines to detect specific proteins in a sample of tissue homogenate or extract. Phosphospecific monoclonal antibodies against each erk. Leandro, ca, and digitizing the results using ivision software. Pathway analysis with the incell western assay learn more about incell westerns. Each software package utilizes slightly different methods for quantification. We performed western blot analysis on the total lysate protein to assess the changes in erk1 and erk2 protein and their phosphorylation.
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